The myotonias and periodic paralyses are heritable diseases of skeletal muscle in which mutations of voltage-gated ion channels alter the electrical excitability of the fiber. The long-term goals of this project are to characterize the functional defects of mutant channels in these disorders and to determine how abnormal channel behavior produces symptoms in affected individuals. In these disorders, muscle dysfunction is caused by intermittent derangements in the electrical excitability of the fiber, which may be pathologically enhanced or depressed. Myotonia is a disorder of enhanced excitability wherein a single stimulus elicits a high- frequency burst of action potentials that produces involuntary persistent muscle contraction lasting seconds. Conversely, periodic paralysis results from a depolarization -induced loss of muscle excitability. Missense mutations in the adult skeletal muscle sodium channel (NaV1.4) may cause myotonia, periodic paralysis, or a combination of both in the same individual. The pathophysiological basis for this variation in clinical phenotype, all arising from mutations in a common sodium channel gene is a major focus of the studies in this proposal. Our experimental approach is to identify alterations in the behavior of mutant channels by measuring ionic current, and then use computer or animal-based models to explore how specific alterations in channel function give rise to myotonia or periodic paralysis. Aim 1 is to characterize the gating behavior of NaV1.4 channels, with a new focus on characterizing these properties for channels expressed in their native skeletal muscle environment. The availability of two mouse lines generated in our lab with knock-in point mutations in NaV1.4 (M1592V and R669H) offers a unique opportunity to characterize mutant channel behavior as occurs in muscle. Our studies on gating of disease- associated mutations of NaV1.4 will also explore the exciting new finding that missense mutations of arginines within S4 voltage-sensor domains may give rise to gating pore currents through an alternative permeation pathway different from the central pore. The propagation of action potentials into the transverse tubular system (TTS) and the activity-dependent accumulation of K+ therein are critical determinants of susceptibility to myotonia. Aim 2 will provide greater understanding for this important feature of muscle excitability by using state-of-the-art optical methods to measure TTS voltage transients and analytical models to estimate K+ accumulation both in normal mammalian muscle and for mouse models of myotonia and periodic paralysis. Aim 3 is a comparative analysis of the clinical phenotypes and electrophysiological properties of muscle from mice harboring either the M1592V or R669H mutations, as a model for gaining further insight on the mechanistic basis for the divergent phenotypes observed in humans for these allelic disorders of NaV1.4 (hyperkalemic periodic paralysis with myotonia contrasted by hypokalemic periodic paralysis without myotonia). PUBLIC HEALTH RELEVANCE: The proposed studies are designed to provide a more complete understanding of the pathophysiologic basis for a group of human neuromuscular diseases: from gene defect to clinical symptoms. These studies will also further our knowledge of Na channel function at the molecular level, will lead to an improved understanding of the determinants of muscle excitability, will provide mechanistic insights for a more rational design of therapeutic strategies, and will serve as a model system for understanding other disorders of excitability such as epilepsy, migraine, or cardiac arrhythmia.